Jun 20, 1988 - Department of Botany, University of Georgia, Athens, Georgia 30602 (R.F.S); Department of ... activation state of ribulose-1,5-bisphosphate carboxylase (rub- .... ducted at a Ca of 310 ,bar, except whenthe Ca was modified.
Plant Physiol. (1989) 89, 590-596 0032-0889/89/89/0590/07/$01 .00/0
Received for publication June 20, 1988 and in revised form September 26, 1988
Acclimation of Photosynthesis to Elevated CO2 in Five C3 Species1 Rowan F. Sage*, Thomas D. Sharkey, and Jeffrey R. Seemann Department of Botany, University of Georgia, Athens, Georgia 30602 (R.F.S); Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 (T.D.S); and Department of Biochemistry, University of Nevada, Reno, Nevada 89506 (J.R.S.) ABSTRACT
the capacity of the light-harvesting and electron transport systems to regenerate RuBP (referred to here as a RuBPregeneration limitation on A) limit A at high CO2 ( 22, 29). Short-term exposure to elevated CO2 causes the activation state of rubisco to decline, regardless of whether Pi regeneration or RuBP regeneration serve as the major limitation on photosynthesis (19, 28). When the capacity to regenerate Pi limits A, the rate of electron transport also declines (24). These responses to elevated CO2 reflect regulatory control by the photosynthetic apparatus, which serves to coordinate the rates of RuBP production and consumption, and the rate of triose phosphate production and utilization (19, 24, 27). Despite our understanding of the short-term response of A to elevated C02, little is known about the mechanisms by which photosynthesis responds to long-term (days to weeks) CO2 enhancement. While over the short-term, CO2 enhancement generally stimulates A, long-term exposure is often inhibitory (4, 12, 14, 20, 30, 31). The nature of the inhibition is unknown. Excessive carbohydrate loading of leaves and physical distortion of the chloroplast by enlargement of starch grains are commonly observed in plants grown at high partial pressures of C02 (3, 4, 12), leading to the suggestion that the decline in A at high CO2 results from either feedback inhibitions or physical damage to the chloroplast (4). Alternatively, Bloom et al. (2) and Field and Mooney (7) have suggested that plants maximize resource-use efficiency by allocating resources, principally N, to maintain a balance between all components of the photosynthetic apparatus and between photosynthetic and non-photosynthetic processes. This implies that proportionally more N will be transferred from nonlimiting processes to those processes which limit A in the period shortly after CO2 enhancement. Thus, reduced A may reflect either a reallocation of N away from rubisco and into light harvesting, electron transport and Pi-regeneration processes or reallocation of N from photosynthetic to nonphotosynthetic processes. Since rubisco constitutes the single largest sink for N in the photosynthetic apparatus (18, 21), changes in its content will have the greatest effect on N partitioning within the leaf. Rubisco activity does decline following long-term exposure to high CO2 (14, 15, 30, 31), but it is unclear whether this results from reduced quantity of rubisco protein or a decrease in the activity of existing enzyme (as occurs when the enzyme deactivates or the endogenous inhibitor of rubisco, 2-carboxyarabinitol 1-phosphate binds to the active site) (9). In addi-
The effect of long-term (weeks to months) CO2 enhancement on (a) the gas-exchange characteristics, (b) the content and activation state of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) leaf nitrogen, chlorophyll, and dry weight per area were studied in five C3 species (Chenopodium album, Phaseolus
vulgaris, Solanum tuberosum, Solanum melongena, and Brassica oleracea) grown at CO2 partial pressures of 300 or 900 to 1000 microbars. Long-term exposure to elevated CO2 affected the CO2 response of photosynthesis in one of three ways: (a) the initial slope of the CO2 response was unaffected, but the photosynthetic rate at high CO2 increased (S. tuberosum); (b) the initial slope decreased but the C02-saturated rate of photosynthesis was little affected (C. album, P. vulgaris); (c) both the initial slope and the C02-saturated rate of photosynthesis decreased (B. oleracea, S. melongena). In all five species, growth at high CO2 increased the extent to which photosynthesis was stimulated following a decrease in the partial pressure of 02 or an increase in measurement CO2 above 600 microbars. This stimulation indicates that a limitation on photosynthesis by the capacity to regenerate orthophosphate was reduced or absent after acclimation to high CO2. Leaf nitrogen per area either increased (S. tuberosum, S. melongena) or was little changed by CO2 enhancement. The content of rubisco was lower in only two of the five species, yet its activation state was 19% to 48% lower in all five species following longterm exposure to high CO2. These results indicate that during growth in C02-enriched air, leaf rubisco content remains in excess of that required to support the observed photosynthetic rates.
During short-term (minutes to hours) exposure to elevated partial pressure of C02, the rate of light-saturated CO2 assimilation (A2) in many C3 plants is primarily limited by the capacity to regenerate Pi from phosphorylated photosynthetic intermediates (10, 17, 22, 23, 26). A Pi-regeneration limitation of photosynthesis is characterized by a lack of sensitivity of A to changes in ambient partial pressure of CO2 and/ or 02 (10, 1 1, 17, 22, 23). In contrast, at subsaturating light intensity, ' Research supported by U.S. Department of Energy contract DEFGO8-84ER13234 to T. D. S. and National Science Foundation grant DMB 86-08004 and U.S. Department of Agriculture grant 87CRCR-1-2470 to J. R. S. 2 Abbreviations: A, net rate of CO2 fixation; Ca, ambient partial pressure of C02; Ci, intercellular partial pressure of C02; Na, nitrogen per unit area; RuBP, ribulose 1,5-biphosphate; rubisco, RuBP carboxylase (EC 4.1.1.39); Pi, orthophosphate. 590
ACCLIMATION OF PHOTOSYNTHESIS TO ELEVATED CO2
tion, it is not known whether the relative capacity to regenerate Pi increases in plants grown at high CO2 or whether it declines, as may be the case if carbohydrate loading leads to an increased feedback limitation. In this work, the mechanisms determining the response of A to long-term CO2 enhancement were examined in five C3 dicots. In addition to a gas-exchange analysis of photosynthesis, leaf N, Chl content, and the activity, amount and activation state of rubisco were analyzed in order to partially describe how the leaf physiology responds to elevated CO2. MATERIALS AND METHODS
Growth Conditions
Five C3 species-Phaseolus vulgaris L. cv Linden (kidney bean), Solanum tuberosum L. (potato), Chenopodium album L. (lambsquarters), Brassica oleracea L. (cabbage), and Solanum melongena L. (eggplant)- were grown in Reno, NV, from April through July 1987 in greenhouses at normal (300 ,ubar in Reno) and high (900-1000 ,ubar) ambient partial pressure of CO2. Plants were grown in a sand-loam mixture at 25° to 30°C day (20°C night) and were watered daily with full strength Hoagland solution (8). During the period of study, light intensities of 1600 to 1800 ,Amol m-2 s-' predominated inside the greenhouses. All measurements were conducted on plants 3 to 8 weeks old. In order to study the time course for the response to elevated C02, 3-week-old P. vulgaris plants initially grown at low CO2 were transferred to high C02, and leaf properties were measured during the subsequent 28 d. High-CO2 partial pressures in the greenhouse were established by manually bleeding in pure CO2 (source: Cardox Corp., Walnut Creek, CA) at a rate required to maintain 900 to 1000 ubar. Ambient CO2 levels were monitored with a Binos infrared gas analyzer (Leybold-Heraus, Hanau, FRG) in the absolute mode. Contamination of the greenhouse atmosphere by sulfur and N containing gases was minor, estimated to be less than 10 parts per billion (by volume). No ethylene was detected in the gas source. Gas-Exchange Measurements All gas-exchange measurements were conducted at saturating light intensities (1100-1800 gmol m-2 s-', depending on species). The CO2 and temperature responses of photosynthesis were determined for fully expanded leaves at high (180 mbar) and low (30 mbar) partial pressure of 02 using a nullbalance gas-exchange system previously described (17). C02response curves were measured at 180 mbar O2 by first placing leaves in an environmentally controlled cuvette at the growth Ca and a water vapor pressure deficit between leaf and air of 6 to 10 mbar. Following a 20 min period for equilibration, the Ca was reduced to 80 ,ubar and then increased in steps to a maximum value, with measurements being made at each step. Occasionally, at the highest C. used, the partial pressure of 02 was reduced to 30 mbar so that the sensitivity of A to 02 could be determined. The temperature response of photosynthesis at high and low partial pressure of 02 was determined in order to calculate
591
the 02 sensitivity of photosynthesis. Measurements were conducted at a Ca of 310 ,bar, except when the Ca was modified so that the Ci was relatively constant at high and low 02. Generally, measurements were first conducted at 24°C, 180 mbar 02. Then the partial pressure of 02 was reduced to 30 mbar and the measurement recorded after A stabilized. The partial pressure of 02 was then returned to 180 mbar. After A was rechecked, the temperature was reduced 4°C and the process repeated. Measurements were conducted at 24°, 20°, 160, 12°C and occasionally 280, 10°, and 8°C. The sensitivity of A to 02 was calculated as 1 - A at 180 mbar 02 divided by A at 30 mbar O2. All gas-exchange measurements were replicated at least twice and usually three times. Gas-exchange parameters were calculated according to von Caemmerer and Farquhar (29). Biochemical Measurements All samples were collected from fully illuminated leaves in the greenhouse at the growth Ca between 9 A.M. and 2 P.M. local time. For rubisco and Chl assays, leaves were sampled by freezing 7 cm2 discs with a hand-operated clamp which consisted of cylindrical copper heads (cooled to liquid N2 temperature) attached to stainless steel tongs. Samples were stored in liquid N2 until analysis. The activity of rubisco was measured as previously described (19) by determining the amount of '4CO2 incorporated into acid-stable products. The activation state of rubisco was determined by comparing the initial activity of rubisco in rapidly ground leaf extracts with the activity of the same extracts which had been incubated 10 to 15 min with 10 mM MgCl2 and 10 mM NaHCO3 at 23°C. The content of rubisco was measured by allowing ['4C]carboxyarabinitol bisphosphate to bind to the catalytic sites as described by Sage et al. ( 19). Leaf Chl content was determined on 200 gL aliquots of the crude leaf extract by adding 800 ,L acetone and measuring the absorbance at 645 and 663 nm. Leaf N was determined on a separate set of leaf discs from those used for rubisco analysis. Leaf discs were collected with a hole punch, dried at 70°C, and N content was determined using a modified microKjeldahl digestion and a Technicon (Tarrytown, NY) autoanalyzer (21). Leaf dry weight per area was determined on the same discs used for N analysis. Where appropriate, differences between treatment means were tested using a pooled-variance Student's t-test.
RESULTS
CO2 Response of Photosynthesis According to the photosynthesis model of von Caemmerer and Farquhar (29) as modified by Sharkey (22), the initial slope of the CO2 response of photosynthesis reflects the number of catalytically competent active sites of rubisco. At partial pressures of CO2 above the initial slope region, A is determined by the capacity to regenerate either Pi or RuBP. Photosynthesis which is limited by the capacity for RuBP regeneration is stimulated by increasing CO2 up to very high C1 (>1000 gbar) and is sensitive to changes in the partial pressure of 02. Only above very high Ci (>1000 ubar) should
the RuBP-regeneration-limited rate of photosynthesis exhibit CO2 saturation. In contrast, photosynthesis which is limited by the capacity for Pi regeneration is little affected, and may be inhibited, by increasing CO2 or decreasing 02 (22). In Figure 1, the CO2 response of photosynthesis is presented for four of the five species studied. These curves are representative of two to three separate C02-response curves determined for each species at each treatment. At the respective growth Ca, the Ci in plants grown at low CO2 was 200 to 250 ,ubar; the Ci for plants grown at high CO2 ranged from 620 to 850,ubar (Fig. 1). In C. album (Fig. IA), plants grown at high CO2 exhibited a lower initial slope than those grown at low C02, indicating that the rubisco capacity had been reduced. Increases in the measurement Ci above 400 gbar reduced A in plants grown at low C02. Lowering 02 from 180 to 20 mbar also reduced A (triangles, Fig. IA). In contrast, in plants grown at high C02, A was stimulated by increasing Ci up to 900 Abar and by reducing the partial pressure of 02, indicating that plants grown at high CO2 had a relatively greater Pi-regeneration capacity. As a result, above 600 ytbar, plants grown at high CO2 consistently had a slightly greater C02-assimilation rate than plants grown at low CO2. In P. vulgaris, plants grown at high CO2 exhibited a lower initial slope than plants grown at low C02, yet the C02saturated rates of photosynthesis were similar (Fig. 1B). Plants grown at high CO2 also exhibited a higher C02-saturation point and greater CO2 sensitivity above a Ci of 400 ,abar than plants grown at low CO2. This response to high growth CO2 is similar to that previously observed in P. vulgaris by von 50
(I
Plant Physiol. Vol. 89, 1989
SAGE ET AL.
592
40
N
30 E
20
0
10
E 0
40
Caemmerer and Farquhar (30). In the other species, growth at elevated CO2 led to either a reduction in both the initial slope and the rate of A at high CO2 (B. oleracea and S. melongena; Figs. 1C and not shown), or a stimulation of C02saturated A with no effect on the initial slope (S. tuberosum; Fig. ID). In S. tuberosum plants grown at high C02, A was sensitive to increasing Ci above 600 ,bar, in contrast to the response of plants grown at low CO2. Similarly, in S. melongena, plants grown at high CO2 exhibited a greater CO2 sensitivity of A above 600 ,bar than plants grown at low CO2 (data not shown). Oxygen Sensitivity of Photosynthesis versus Temperature
As temperature declines, a Pi-regeneration limitation becomes more pronounced in many species, eventually limiting A at a Ci of 250 ubar and ambient partial pressure of 02 (17, 23). This causes the 02 sensitivity of A to decline until at a given temperature, no change in A is observed when the partial pressure of 02 is reduced from 180 to 30 mbar (17).
The temperature at which A is completely insensitive to the partial pressure of 02 is a useful index of the relative capacity of plants to regenerate Pi. Plants with a lower Pi-regeneration capacity will exhibit no 02 sensitivity at warmer temperatures than those with a relatively higher Pi-regeneration capacity. In addition, at a given temperature above the point of zero sensitivity, plants with a lower Pi-regeneration capacity will have lower 02 sensitivity. The temperature response of A at 180 and 30 mbar O2 iS shown for C. album only (Fig. 2). In Figure 3, the temperature response of the O2 sensitivity of A is shown for 4 of the 5 species. Below 22°C, plants grown at high CO2 had a greater 02 sensitivity of A than plants grown at low CO2 (Figs. 2 and 3). In individuals of C. album grown in high C02, A lost 02 sensitivity near 12°C, about 6°C lower than plants grown at normal CO2 levels. These data indicate that acclimation to high CO2 involves at least a partial removal of a Pi regeneration limitation on A. This conclusion is further supported when measured 02 sensitivities are compared with modelled sensitivities calculated according to Sage and Sharkey (17) assuming that either rubisco or RuBP regeneration limit A 60
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Intercellular C02, ,ubar CD
Figure 1. C02 response of photosynthesis in plants grown at an ambient CO2 partial pressure of 300 Abar (filled symbols) or 900 to 1000 libar (open symbols). The arrows indicate the intercellular partial pressure of C02 measured when the ambient C02 was set equal to the partial pressure of CO2 at which the plants were grown. Measurements were conducted at a partial pressure of 180 mbar 02 except for triangles in A, which represent measurements at 30 mbar 02. Note that the open triangle at a Ci of 500 ,bar is partially obscured.
lC
15
20
25
10
15
20
25
30
Leaf temperature, 0C
Figure 2. Temperature response of photosynthesis in C. album at 02 partial pressure of either 180 (0) or 30 (0) mbar. At 180 mbar 02, leaves from both treatments were measured at an ambient partial pressure of C02 (C.) of 310 jAbar. At 30 mbar 02, leaves from both treatments were measured at an ambient C02 of 340 gbar. an
ACCLIMATION OF PHOTOSYNTHESIS TO ELEVATED C02
(Fig. 3A). Except at the lowest measurement temperatures, measured and modelled 02 sensitivities are similar in plants grown at high CO2 in all four species. In plants grown at low C02, measured sensitivities are generally below modeled sensitivities at all but the highest measurement temperatures.
Table I. Leaf Dry Weight Per Area (Lv), Nitrogen Content Per Area (N.) and Chl Content of Leaves from C. album, S. tuberosum, S. melongena, and B. oleracea Grown at Either 300 or 950 (± 50) Mlbar
CO2a Species
Leaf Weight per Area, Leaf N, and Leaf Chl In all species, growth at high CO2 significantly increased leaf dry weight per area (Table I, Fig. 4). In P. vulgaris, large increases in leaf weight per area were observed between the first and third week following transfer of plants from low to high CO2 (Fig. 4). Much of the increase in leaf weight per area was probably due to the accumulation of starch (3, 12). Following long-term exposure to high CO2, leaf N content per unit area (Na) increased in the two Solanum species, but was little changed in C. album, P. vulgaris, and B. oleracea (Table I, Fig. 4). N per unit weight fell in all species following exposure to high CO2 (data not shown), but this was largely a consequence of the increase in leaf weight per area and does not necessarily reflect a change in the amount of N allocated for light and CO2 capture. As with leaf Na, there was no consistent pattern in the response of leaf Chl. Growth at high CO2 led to a decline in Chl content in C. album and B. oleracea, but had little effect on Chl content in the other three species (Table I, Fig. 4).
Rubisco Content and Activation State
In B. oleracea and to a lesser extent in C. album, leaf rubisco content was lower in plants grown at high CO2. In the other three species, rubisco contents were similar after 3 to 8 C. album
A
P. vulgaris
0
40 Responses Q I-, 30 Modelled RuBP lmited° Rubisco
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Figure 3. Temperature response of the 02 sensitivity of photosynthesis in plants grown at an ambient C02 partial pressure of 300 (@) or 900 to 1000 ybar (0). % 0 sensitivity equals (1 A at 180 mbar 0 divided by A at 30 mbar 02)x1 00%. Ambient C02 was 310 Abar at 180 mbar O2 and 340 /tbar at 30 mbar O2. Intercellular C02 partial pressures were similar at 180 and 30 mbar O2, but ranged from 220 Abar at the warmer temperatures to 280 ,tbar at the cooler temperatures. The modeled responses in Figure 3A were calculated according to Sage and Sharkey (17) assuming an intercellular C02 partial pressure of 240 Abar and the C02-assimilation rate to be limited by either the RuBP regeneration or rubisco capacity. -
593
C. album
S. tuberosum
S. melongena
B. oleracea
300
950 9 m-2
55a ±7
95b ±5
Na
Chl
300 950 mmolm 2
300 950 pMoI m-2
133 ±12
158 ±8
(4)
739a 633b ±15 ±15
(4)
(4)
(4)
(6)
(6)
40a ±1
64b ±2
107a 123b 467 ±4 ±6 ±10
500 ±28
(5)
(6)
(5)
(6)
(5)
(7)
55a 84b ±2 ±4 (3) (3) 80a 138b ±4 ±6
108a 146b 518 584 ±5 ±8 ±24 ±84 (3) (3) (4) (3) 113 85 367a 240b ±21 ±2 ±22 ±9
(3)
(3)
(3)
(3)
(5)
(6)
are given as means ± SE. Numbers in parentheses are sample sizes. Letters indicate statistically different populations at P b Growth C02, 300 or 950 (±50) ubar. < 0.05. a Results
weeks exposure to high CO2 (Table II, Fig 4). During the first 3 weeks following transfer to high CO2, rubisco content increased in leaves of P. vulgaris grown at both high and low CO2, but at a slower rate in plants grown at high CO2 (Fig. 4). However, by 3 to 4 weeks after the transfer, rubisco contents were similar between the treatments. The percent of leaf N invested in rubisco was lower in plants grown at high CO2, particularly C. album (Table II). Differences in the percent of N invested in rubisco were apparent within a few days after transferring P. vulgaris plants to high CO2 (Fig. 4). Plants grown at low CO2 had a greater (C. album, B. oleracea) or equivalent (P. vulgaris, S. tuberosum, S. melongena) rubisco to Chl ratio relative to plants grown at high CO2 (Table II, Fig. 4). The rubisco activation state, which is believed to reflect the carbamylation state of the enzyme (13), was significantly lower in leaves of all five species when grown at high C02 (Table II, Fig. 5). The activation state of rubisco in Phaseolus vulgaris declined within 1 h after transferring plants to high CO2 and remained low throughout the subsequent 4-week observation period (Fig. 5). The relative difference in the activation state of rubisco between plants grown at high and low CO2 remained constant throughout this period, although the absolute activation state did vary (probably due to differences in the assay, rather than the plants). The catalytic turnover rate (kcat) of fully activated rubisco was unaffected by high growth CO2 (data not shown), indicating that the endogenous inhibitor of rubisco, 2-carboxyarabinitol 1-phosphate, is not responsible for a reduction of rubisco activity at high C02. DISCUSSION At light saturation, atmospheric CO2, and growth regimes similar to those encountered in the field, photosynthesis can
Plant Physiol. Vol. 89, 1989
SAGE ET AL.
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a
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E VI
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Figure 4. Effect of C02 enhancement on (A) leaf weight per area, (B) leaf nitrogen per area, (C) leaf Chl per area, (D) rubisco content per area, (E) rubisco content per nitrogen, and (F) rubisco content per Chi in equal aged P. vulgaris plants following transfer from low to high partial pressure of C02. Low C02 controls (0); high C02 treatment (0). Mean ± SE (except in E, which are means only). In this and Figure 5, sample size per mean varied from four to seven replicate measurements.
'a
Time after transfer, days
Table 11. Properties of Rubisco in Leaves of Four C3 Species Grown at Either 300 or 950 (±50) gbar C028 Rubisco Content
Species
Rubisco Content 300b 950 2 g-m
2.28b ±0.04
C. album
2.97a ±0.13
(5)
(6)
S. tuberosum
2.43 ±0.15
2.33 ±0.20
(7)
(6)
1.90 ±0.10
1.90 ±0.34
S. melongena
(4) B. oleracea
(4) 0.90b
Rubisco
Rioh
Content 300 950 % leaf N
25.5
16.5
25.9
21.7
20.1
Rubisco/Chl 300 950 g
mmoh1
4.Oa ±0.3
State 950
300 %
3.5b ±0.1
89.7a ±3.7
65.6b ±2.7
(5)
(6)
(5)
(6)
5.2 ±0.2
4.9 ±0.5
85.0a
66.3b ±0.9
(7)
(5)
(6)
(6)
14.9
3.7 ±0.2
3.4
12.0
±0.7 (3) 3.5b ±0.4
88.9a ±9 (4) 81.2a ±6.2
41.5b ±5 (4) 61.2b ±4.4
(6)
(5)
(6)
1.56a ±0.27
±0.08
(4) 5.Oa ±0.7
(5)
(6)
(4)
15.8
Rubisco Activation
±1.7
Values are means ± SE. Numbers in parentheses are sample sizes. Letters indicate statistically b Growth C02, 300 or 950 (±50) Abar. different populations at P < 0.05. a
be simultaneously limited by both the capacity of RuBP regeneration and rubisco activity (6, 29, 30). Short-term CO2 enhancement disrupts the balance between rubisco, RuBP regeneration, and possibly Pi regeneration. In response, the capacity of processes which are non-limiting at high CO2 (RuBP carboxylation and possibly RuBP regeneration) are regulated downward so that they remain balanced with the limiting processes (either Pi or RuBP regeneration) (19, 24). If a plant exhibits perfect acclimation, then it should reallocate N and other resources away from the down-regulated, nonlimiting components to the limiting components. As the capacities of the limiting and nonlimiting processes are realigned, the regulation on the nonlimiting enzymes should be relieved, allowing them to operate at full capacity. With respect to rubisco, this implies that after transfer to C02-enriched air, its activation state should recover as the excess rubisco enzyme
is degraded. If the leaf does not fully acclimate, then rubisco will remain partially deactivated. In this way, the rubisco activation state may be an effective indicator of the acclimation potential of the photosynthetic apparatus. In this study, acclimation to high CO2 did not involve a recovery of the activation state of rubisco. In all species, the rubisco activation state was reduced after prolonged exposure to high CO2, and there was no relationship between a change in rubisco content and its activation state. This is well documented in P. vulgaris, where the activation state declined immediately after transfer to high CO2 and remained low throughout the subsequent four-week exposure period (Fig. 5). In three of the five species (P. vulgaris, S. tuberosum, and S. melongena) there was little evidence that rubisco played any role in the acclimation process. Leaf rubisco contents differed little between treatments as did the rubisco to Chl
ACCLIMATION OF PHOTOSYNTHESIS TO ELEVATED C02 0)
4l)
100 U)
CD C:
90
0
80 CI)
1 hr_< 0 -0'
70 0.
r,n t)UI 0
5
10
15 20 25
Time after transfer, days Figure 5. Effect of C02 enhancement on the activation state of rubisco in P. vulgaris following transfer from low to high partial pressure of C02. Low C02 controls (0); high C02 treatment (0). Mean ± SE.
ratio, which is an indicator of the N investment in rubisco the light reactions (5, 21). Only C. album and B. oleracea exhibited lower rubisco contents and reduced rubisco to Chl ratios. However, the lack of recovery of the activation state of rubisco in these two species indicates that the photosynthetic apparatus was not able to fully acclimate to growth at high C02. CO2 enhancement should increase the N-use efficiency of C3 photosynthesis by increasing the rate of carboxylation and decreasing the rate of oxygenase activity per unit photosynthetic protein. However, failure to fully activate rubisco at high CO2 restricts the degree that photosynthetic N-use efficiency is stimulated by CO2 enrichment. For example, at high C02, rubisco contains between 12% and 23% of leaf N in these five species. Because about 30% to 60% of the active sites are nonfunctional in these species at high C02, at least 5% to 9% of the leaf N is not used. In all five species, the Pi-regeneration limitation on A evident in plants grown at low CO2 appeared to be reduced or removed during acclimation to high CO2. Above 400 to 600 gbar Ci, high-CO2-grown plants of four species, C. album, P. vulgaris, S. tuberosum, and S. melongena exhibited a more pronounced stimulation of A by increasing CO2 than plants grown at low CO2. In addition, in these four species, plants grown at high CO2 had a greater C02-saturation point, indicating that the Pi-regeneration capacity increased relative to the RuBP-regeneration capacity. In all five species, oxygen sensitivity was greater below 22°C in plants grown at high CO2. In C. album and S. tuberosum, the increase in the C02saturated rate of A indicated that acclimation to high CO2 involved an increase in the absolute Pi-regeneration capacity. An increased Pi-regeneration rate requires an increased rate of starch and sucrose synthesis. This can occur if the level of starch and sucrose-synthesizing enzymes increase. Sharkey et al. (25), for example, demonstrated that the presence of a Piregeneration limitation in Flaveria linearis is related to low activity of cytosolic fructose- 1 ,6-bisphosphatase. Alternatively, Pi-regeneration limitations may result if Pi levels in the cytoplasm fall below an optimum, causing a depression of sucrose synthesis (10, 11). Acclimation may involve a versus
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reestablishment of the Pi optimum, perhaps by increasing cytoplasmic Pi levels (10). In P. vulgaris and B. oleracea, the Pi-regeneration limitation on A appeared to be removed as a result of a decline in the rubisco and RuBP-regeneration capacities, rather than an increase in the absolute capacity for Pi-regeneration. This pattern may not reflect acclimation but may be a stress response caused by excess starch accumulation and subsequent distortion of the chloroplast. Azcon-Bieto (1) observed that high carbohydrate levels of leaves were associated with reduced photosynthetic rates and CO2 and 02 sensitivity. In contrast, from the data presented here, leaves with the greatest weight per area, and presumably, the highest starch content, exhibited greater degrees of 02 and CO2 sensitivity of photosynthesis than leaves with relatively low leaf weight per area. Similar results have been observed between plants grown at high and low N supply (16). These results demonstrate that conditions leading to starch accumulation are not necessarily associated with a Pi-regeneration limitation. Instead, Pi-regeneration appeared to be more limiting under conditions where starch accumulation was minimal. In two species (C. album and B. oleracea), a decline in rubisco content was associated with a decrease in the initial
slope of the C02-response of photosynthesis. In P. vulgaris, there was no change in rubisco content following long-term exposure to elevated C02, yet the initial slope declined. Deactivation of rubisco will reduce the number of catalytically competent active sites and this can reduce the initial slope if rubisco remains deactivated when measurements are made at low CO2. Following short-term (30 min) exposure to elevated C02, the activation state of rubisco completely recovers after returning to low C02 in P. vulgaris (19). However, preliminary results indicate that following long-term exposure to elevated C02, P. vulgaris plants were unable to reactivate rubisco within 4 h after they were transferred to low CO2 (data not shown). In S. tuberosum, by contrast, neither the initial slope nor the rubisco content differed between treatments, indicating that plants grown at high CO2 were able to completely reactivate rubisco when the Ci was reduced below 300 ,tbar for measurement purposes. In summary, no two species exhibited identical quantitative responses to CO2 enrichment, yet all exhibited reduced rubisco activation states and a reduction in the degree that the Pi-regeneration capacity limited photosynthesis. No species exhibited an idealized acclimation response at the biochemical level, and three of the five species (P. vulgaris, S. melongena, and B. oleracea) may have been negatively affected by growth at high CO2. The weedy species, C. album, exhibited the most economical response to long-term CO2 enrichment because its rubisco content decreased yet the rate of photosynthesis at high CO2 increased. In the high CO2 atmosphere of the next century, these adjustments may enhance its ability to compete against native and agricultural species with less effective acclimation responses. ACKNOWLEDGMENTS We wish to thank Judy Miles for expert laboratory help and Patty Moen for gas-exchange assistance.
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